Most membrane and secreted proteins are decorated with mucin-type O-glycans which serve diverse biological roles. The mechanisms behind their biological roles and more specifically what governs O-glycan site selection and subsequent O-glycan elongation are largely unknown. The objective of this project is to elucidate the processes governing O-glycan site selection and O-glycan elongation at the enzyme and peptide substrate level in order to address the molecular mechanisms and biology of O-glycosylation. Mucin type O- glycosylation is vital to animal reproduction and development and increasingly linked to a wide range of rare to common disease states (such as hormonal/metabolic dysfunction, impaired host defense, inflammatory and cardiovascular diseases and even cancers) where it serves to modulate diverse biological functions including cell-cell interactions. Many disorders are linked to changes in expression or mutation of individual members of the large family (20 in man) of polypeptide-GalNAc transferases (ppGalNAc Ts) that initiate O-glycosylation by adding GalNAc to polypeptide Ser or Thr residues. O-glycosylation is absolutely required for embryonic development of the mouse and fly, relying on the Core 1 elongating transferase (T-synthase) in the mouse and several individual ppGalNAc T isoforms in the fly. How individual ppGalNAc T isoforms in this large family (or even T-synthase) can play such critical biological roles is unknown as the substrate specificity of these transferases have not been sufficiently characterized. That ppGalNAc T site selection is also modulated (positively or negatively) by prior glycosylation underscores its complexity and how much more needs to be learned of its specificity. The detailed characterization of ppGalNAc T isoform and elongating transferase specificity proposed in this project will lead to our understanding of the molecular mechanisms underlying the biological roles of O-glycosylation and will eventually lead to novel strategies to treat diseases of aberrant O- glycosylation. The AIMS of this project are to (1) expand the innovative use of a library of novel random peptide and glycopeptide substrates to fully characterize the specificity and basic enzymology of the transferases that initiate and elongate mucin type O-glycosylation, (2) to use these data to further develop sophisticated web based O-glycan predictive tools that include ppGalNAc T isoform and elongating transferase peptide and glycopeptide specificity, and (3) to develop a novel method for identifying in vivo isoform specific glycosylation targets using tissues from transferase knock-out animal models. These basic studies together with those of colleagues at other institutions will advance our understanding of the properties of these transferases, their targets (and resultant glycan structures) and ultimately the mechanisms of their biological role and function. These studies will significantly advance the field and will allw the development of novel specific inhibitors for potential use as targeted therapeutics.